Traumatic brain injury (TBI) is a complex brain dysfunction caused by an outside force, usually a violent blow to the head and can result in physical, cognitive, social, and behavioral symptoms. A core symptom observed following TBI is increased fear generalization, as defined by the overgeneralization of fear from a conditioned, fear-inducing stimulus to novel, neutral stimuli. Fear generalization can lead to heightened, debilitating anxiety and maladaptive responses in a safe environment. A previous study has shown that mild TBI (mTBI) in rats results in generalized learned fear to both conditioned and novel stimuli. However, the neural ensembles mediating this increased fear generalization have yet to be identified. My laboratory uses a behavioral assay called contextual fear discrimination (CFD) to assess fear generalization in mice. We shock a mouse in one context (context A), and then we can measure whether they can discriminate between this aversive, shock- paired context and a similar, but safe context (context B) over approximately 10 days of discrimination learning. In this grant proposal, I will use this CFD paradigm, in combination with an activity-dependent tagging genetic mouse line to identify and pharmacologically manipulate the neural ensembles that underlie altered fear generalization following TBI. In Aim 1, we will identify and quantify how TBI alters the neural ensembles mediating fear generalization by utilizing the ArcCreERT2 x EYFP mice. This mouse line allows for the indelible labeling of cells expressing the immediate early gene (IEG) Arc/Arg3.1 and allows for a comparison between the cells that are activated during the encoding of a memory and those that are activated during the retrieval of the corresponding memory. Memory recall or expression is mediated by reactivation of the same neurons that were active during memory acquisition. Therefore, using our activity-dependent tagging mouse line, we can determine where and how TBI impacts fear generalization neural ensembles throughout the brain. In Aim 2, we will pharmacologically manipulate sham and TBI mice with the goal of improving behavioral fear overgeneralization and the corresponding neural circuits. Recently, ketamine has emerged as an anesthetic and sedative agent for TBI injuries with promising results. Furthermore, we have preliminary data indicating ketamine is effective at decreasing fear generalization in TBI mice. Here, both male and female mice will be administered a single dose of saline, (R,S)-ketamine, or one of its metabolites immediately after sham or TBI surgery. CFD and ex vivo whole-brain imaging will be utilized in order to determine the effectiveness of these treatments on fear overgeneralization behavior and on the underlying neural ensembles. The premise of this grant proposal is to identify and quantify fear overgeneralization neural ensembles in a mouse model of TBI with the goal of developing novel treatments. The outcome of this targeted rescue will provide direct evidence that disrupted neural ensembles result in fear overgeneralization in a mouse model of TBI. !